Extracellular vesicles (EVs) play an important role in many physiological and pathological processes by participating in intercellular communication. Therefore, the isolation and analysis of EVs have a great value for understanding their biological functions and developing EV-based diagnosis and treatment methods for diseases. The efficient isolation and highly sensitive and reliable detection of EVs largely depend on the recognition ligands. Aptamers are a type of single stranded oligonucleotides that can efficiently and specifically bind to their targets. Their merits of easy modification and programmability make them ideal recognition ligands for EV isolation and analysis. To improve the isolation efficiency of EVs, various strategies have been proposed to enhance the affinity of aptamers and the contact probability between interfaces and EVs. In addition, the isolation of EV subtypes helps to understand the biological significance of EVs. In terms of EV analysis, methods such as electrochemistry, visualization, surface enhanced Raman spectroscopy (SERS) and fluorescence were developed, according to the transduction modes of the recognition signals of aptamers and EVs. This review summarizes recent progress, challenges, and future directions in the selection of aptamers and their applications in EV isolation and analysis.